In recent years, immunochromatographic strip type immunoassay has become more important as a simple in-vitro diagnostic kit or portable diagnostic device for detecting an antigen in a sample solution by making use of the specific reactivity which an antibody has. In particular, simple multiplex analysis tools based on immunochromatography for analyzing the presence or absence of infection with pathogens such as influenza virus or bacteria have been under research and development.
Colloidal metal particles or latex particles have generally been used as an insoluble carrier to be used in an immunological measurement method. Latex particles need a cumbersome production step such as modification of a chemical functional group in order to firmly support a substance such as protein to be labeled. Therefore, colloidal gold particles capable of easily supporting a substance to be labeled and are produced conveniently at a low cost have been preferably used.
Although immunochromatographic test drugs which have labeled an antibody with an insoluble carrier have been used generally since they are easy to operate and need only a short time for the test, a line which can be observed when the test result is positive is not clear since they have generally lower sensitivity in comparison with EIA.
In order to overcome such a problem, various metal colloids having a higher sensitivity than conventional colloidal metal particles already put into practical use and are suitable as labeling agents for immunological measurement or protein staining agents are developed.
Patent Document 1 provides colloidal metal particles having an average particle size of from 50 to 150 nm obtained by supporting platinum on the surface of colloidal metal particles (average particle size: from 30 to 100 nm) since colloidal platinum particles do not develop color sufficiently due to small average particle size and are not suited for practical use in immunochromatography. The colloidal metal particles are prepared by reducing chloroauric acid in a solvent to form colloidal gold particles and then reducing chloroplatinic acid in the presence of the resulting colloidal gold particles (refer to Patent Document 1).
Patent Document 2 provides colloidal metal particles obtained by improving the above-mentioned colloidal metal particles and therefore having higher sensitivity. Namely, colloidal metal particles (average particle size: 30 to 100 nm) having platinum supported thereon which has an average particle size of 5 nm are provided. They are prepared in a production method wherein by adjusting, to a predetermined range, the amount of a reducing agent added in preparing colloidal metal particles in a medium and the amount of a reducing agent added in reducing and supporting platinum on the colloidal metal particles and wherein the medium does not substantially contain a protective colloid forming agent. Examples of such protective colloid forming agent include water soluble high-molecular substances such as PVA, PVP, and gelatin, surfactants, and high-molecular chelating agents (refer to Patent Document 2).
As another method for improving the sensitivity in immunological and immunocytological diagnostic test, a method of coating gold sol ultrafine particles with an alkanethiol (derivative) to impart the gold sol surface with certain hydrophobic-hydrophilic balance so as to prevent aggregation which is caused by a salt by which non-specific interaction between the gold sol surface and an exogenous protein (refer to Patent Document 3) is minimized is provided.
On the other hand, in vitro diagnostics for pregnancy diagnosis, red-colored spherical colloidal gold particles already put on the market have been improved to exhibit higher sensitivity. Colloidal gold is required to have a particle size suited for an intended use; have a sharp particle size distribution; and have a uniformly spherical shape so that a production process of it is under development.
Patent Document 4 includes a nucleus formation stage of adding a first reducing agent (citrate) to a solution of a first gold salt to form colloidal nucleus particles (average particle size: from 12 to 17 nm) and a growth stage of simultaneously adding, to the solution of the colloidal nucleus particles, a second gold salt and a second reducing agent (ascorbate) to grow a colloidal nucleus. This growth stage is conducted at least once. The average particle size of the colloidal gold particles is 17 nm or greater and less than 55 nm in the first growth stage; 55 nm or greater and less than 110 nm in the second growth stage; and from 110 to 220 nm in the third growth stage. The standard deviation of the particle diameter is within 10% (refer to Patent document 4).
In the case of testing only one item such as a pregnancy test kit for finding whether pregnancy or not, it is only necessary to use one labeling agent in visual judgment. Recently, a multiplex test should be conducted when it is necessary to identify a causative virus as in a virus test in cold-like infections or respiratory infections. Thus, various test systems have been developed with a view to easing the burden of patients and health care workers.
For example, although there is a known lateral flow type immunoassay capable of detecting a plurality of viruses (rotavirus, calcivirus, coronavirus, adenovirus, enterovirus, and the like) by using one test tool, the assay has the problem that a plurality of detection lines tend to lead to erroneous visual judgment.
In the test of a virus in respiratory infections by using immunochromatography, a testing method including pretreating a specimen such as nasal discharge, sputum or a swab from the nasal cavity with a specimen treatment solution to prepare a test sample suited for the test of a plurality of respiratory infections and analyzing respective portions of the resulting test sample by using a plurality of test tools such as a first test tool (for example, testing an influenza virus infection) and a second test tool (for example, testing an adenovirus infection or an RS virus infection) (refer to Patent Document 5) has been developed.
Further, a measurement method including immunochromatography having a high ability of judging with labeled antibody particles having an arbitrary color and capable of simultaneously measuring two or more measurement objects by using two or more labeled antibody particles has been developed. More specifically, hCG and LH are measured simultaneously by using a luminescent dye such as TRITC (absorption maximum: about 550 nm, red) and FITC (absorption maximum: about 500 nm, orange) (refer to Patent Document 6).
When multiplex tests are conducted simultaneously through visual judgment by using one test tool and labeling agents or protein staining agents used are of the same color or similarcolor, there is a possibility of causing misjudgment or wrong diagnosis. In order to prevent misjudgment or wrong diagnosis by visual judgment, it is desired to conduct visual judgment by using labeling agents or protein staining agents of highly distinguishable colors.
When two colors are present, their distinguishability differs with the colors used in combination. Since a red color and a blue color can be highly distinguished from each other by visually view, they are used for various distinguishing purposes as can be seen in indications for distinguishing between male and female or indications for distinguishing between hot water (red) and water (blue). Colloidal gold particles which have been conventionally put into practical use are red-colored spherical particles. If blue-colored colloidal gold particles different in color, in other words, highly distinguishable from red color are used as a labeling agent or protein staining agent, misjudgment or wrong diagnosis through visual judgment is presumed to decrease markedly. However, blue-colored colloidal gold particles have not yet been put into practical use.
In patent Documents 7 to 9, metal nanoparticles having light absorption wavelength properties varied by changing the size, pattern, structure/shape or the like of metal nanoparticles are described.
According to Patent Documents 7 and 8, blue-colored gold nanoparticles have a structure/shape of gold nanoshells, nanorods, nanotubes, or nanoprism particles; the gold nanoparticles are produced by (1) adding a reducing agent to a yellow-colored silver nanoparticle solution (containing a protecting agent such as polyvinylpyrrolidone or ethylene glycol) and then, refluxing the resulting mixture at about 100° C., (2) pouring a gold salt solution in the reaction mixture thus refluxed to react them, and (3) after cooling to normal temperature, the reaction mixture is filtered through a 0.2 μm microfilter; and the gold nanoparticles thus obtained are made of gold (gold nanoshell) only at the surface layer thereof. According to these documents, gold nanorods, gold nanotubes, or gold nanoprisms are obtained by using a surfactant such as hexadecyltrimethylammonium bromide (bromide) (C6TAB) in formation of gold nanoparticles. These documents do not include a definite description on the size of the particles. They include a description on the use of them as pigment for cosmetics but do not include a description on the use of them as a labeling agent or protein staining agent in immunoassay (refer to Patent Documents 7 and 8).
Patent Document 9 describes rod-like gold nanoparticles obtained by reducing a gold ion with a reducing agent (an amine) in an aqueous solution containing Cl6TAB (a surfactant of an ammonium salt). The aspect ratio (long axis/short axis) of the gold nanoparticles can be controlled by regulating a mixing ratio of the amine and the ammonium salt used in combination. By doing so, gold nanorods having an aspect ratio of from 2 to 11 and an absorption wavelength peak area of from 658 to 1200 nm are obtained. According to the description, these gold nanorods can be used as a test drug (refer to Patent Document 9).
Since the gold nanoparticles thus obtained contain C16TAB as a surfactant, they are not suited for directly supporting (modifying) with protein such as detection antibody. Since it needs a cumbersome operation such as removal or substitution of the surfactant, it is not preferred as a labeling substance for a protein to be used as a test drug in the immunological measurement method. In addition, they are not preferred from the standpoint of handling because C6TAB has toxicity.
Non-patent Document 1 describes a colloid of stick-shaped gold nanocrystals exhibiting a bluish green color. The stick-shaped gold naocrystals have a complex three-dimensional structure; have from one to eight protrusions; and have a crystal size, including the protrusion, of from 30 to 50 nm (protrusion length of from about 15 to 25 nm and a width of about 8 nm). The three-dimensional branch-shaped gold nanocrystals are obtained in a high yield (92%) by reacting an aqueous solution of chloroauric acid and an organic acid (HEPES, HEPPSO, PIPES, or the like) which is a Good's buffer component at room temperature (refer to Non-patent Document 1).
However, the colloid of branch-shaped nanocrystals obtained in Non-patent Document 1 and exhibiting a bluish green color has a crystal size of from 30 to 50 nm, which is not a desired size. Therefore, even if it is used as an immunochromatographic diagnostic agent, insufficient color development prevents smooth visual judgment.
As can be seen in the related art documents, a colloid of gold nanocrystals exhibiting a bluish green color is not suited as a labeling carrier of an immunochromatographic diagnostic agent since the colloidal particle size is as relatively small as from about 30 to 50 nm. In addition, so-called multipod-shaped, branch-shaped, or confeito-shaped ones often use a shape stabilizer and the shape stabilizer makes it difficult to achieve direct modification of gold nanoparticles with a protein.